The present invention relates to the use of peptide conjugate compositions as an immunogen, with each peptide conjugate contained therein comprising a target antigenic site on the third constant domain (CH3) of the to epsilon (xcex5) heavy chain of IgE, with said target antigenic site covalently linked to (1) a carrier protein through chemical coupling, or (2) a helper T cell epitope and other immunostimulatory sequences through chemical coupling or through direct synthesis, for the treatment of allergy.
More particularly, the present invention relates to the use of such peptide conjugate compositions as an immunogen to elicit the production, in mammals including humans, of high titer polyclonal antibodies specific to a target effector site on the CH3 domain of the xcex5 heavy chain of IgE, and to the use of such composition as a pharmaceutical to provide an immunotherapy for IgE-mediated allergic diseases.
In the immune system of humans and other mammals, IgE mediates type I hypersensitivities. These are the allergic responses to certain foods, drugs, and environmental allergens which are manifested by such symptoms as allergic rhinitis, asthma, allergic dermatitis, and anaphylaxis. Existing strategies to treat allergic diseases are of limited utility, consisting of attempts to either desensitize the atopic individual to an identified allergen or to ameliorate an ongoing allergic reaction with therapeutic compounds. Limitations to allergen-based desensitization immunotherapy include difficulties in identifying the allergen involved and the adverse reactions frequently caused by the use of the identified allergen (World Health Organization and International Union of Immunological Societies Working Group, Lancet, 1989; i:259-261). Other treatments for the relief of allergies employ therapeutic compounds to block the acute inflammatory cascade that is responsible for allergic reactions. These compounds include anti-histamines, decongestants, xcex22 agonists, and corticosteroids. Anti-histamines, decongestants, and xcex22 agonists act on events downstream of IgE in the allergic cascade, making them palliative remedies which address allergic symptoms rather than preventative treatments which must act on events closer to the initiation of IgE-mediated allergic reactions. These palliative remedies provide relief that is short term and partial, frequently accompanied by adverse side effects. Many patients with severe allergies are effectively treated with corticosteroids. Steroid therapy reduces inflammation but is broadly immunosuppressive.
To avoid the shortcomings of the known therapeutic drugs, it would be more desirable to prevent allergic responses by selective intervention targeted to IgE. In common with the other immunoglobulins, IgE has two heavy chains and two light chains. The xcex5 heavy chain has five domains, a variable VH domain and constant domains CH1 to CH4. The constant domains from both xcex5 chains of an IgE molecule combine to comprise the constant or Fc region of IgE. IgE circulates and becomes attached to effector cells such as basophils and mast cells through a site on the IgE Fc region, becoming bound to a high affinity Fcxcex5RI receptor on the cell surface. In an allergic response, allergens (e.g., pollen, dust mite proteins, flea antigens) bind to the antigen-binding sites on the variable region of mast cell or basophil-bound IgE. This action crosslinks the IgE molecules and the underlying Fcxcex5RI receptors. The IgE-allergen complexes thereby signal the degranulation of mast cells and basophils with the concomitant release of histamine and the other inflammatory mediators. These mediators produce the symptoms of allergy, up-regulate the production of IgE, and result in heightened sensitivity to the allergen (Davis et al., Springer Semin Immunopathol, 1993; 15: 51-73).
It has been suggested that allergic diseases may be treated by interventions which inhibit the binding of IgE to mast cells and basophils. For example, synthetic peptides corresponding to various sites on the Fc of IgE have been studied as competitive inhibitors for the binding of IgE to the Fcxcex5RI receptor, The presumption of the investigators has been that such peptides act as antagonists for sites on IgE that participate in the binding of IgE to the Fcxcex5RI receptor, and serve to map portions of the binding site.
The amino acid residues of the competitively inhibiting IgE peptides and of all IgE peptides to follow, including non-human IgE peptide homologues, are indexed in accordance with the numbering for human IgE given by Dorrington and Bennich, (Immunol Rev, 1978; 41: 3-25, also accessible at internet location http:/www.pdb.bnl.gov/pdb.bin/pdbids). That human sequence is listed here as SEQ ID NO:1 and is numbered as shown in Table 1. The homologous dog, rat and mouse sequences for IgE (Patel et al., Immunogenetics, 1995; 41: 282-286; Steen et al., J Mol Biol, 1984; 177: 19-32; and, Ishida et al., EMBO, 1982; 1: 1117-1123) are also shown in Table 1 and listed as SEQ ID NOS: 2, 3, and 4 respectively. The animal sequences are shown in register with human IgE. Individual amino acid positions in human IgE, and in homologues from other species, are identified herein according to the numbering system for the amino acid sequences shown in Table 1, unless otherwise specified.
Helm et al. (Nature, 1988; 331:180-183) have shown that a 76 amino acid long recombinant polypeptide, spanning the C-terminal CH2 and N-terminal CH3 region of human IgE, from amino acids 301-376, reduces binding of IgE to human mast cells by competitive inhibition. Other studies reported that only the CH3 domain is involved with binding to Fcxcex5RI. For example, a rat sequence peptide corresponding to amino acids 401-415 of the human sequence (Table 1) inhibited the binding of rat IgE to rat mast cells (Burt and Stanworth, Eur J Immunol, 1987; 17:437-440). A peptide of residues 419 to 463 from human IgE prevented the sensitization of rat mast cells (Nio et al., FEBS Lett, 1992; 314: 229-231). Jardieu and Presta (WO 93/04173) reported on peptides homologous to the CH3 and CH4 regions which may include amino acids 373-390, 420-428, 446-453, and adjacent regions, which differentially bind to the Fcxcex5RI receptor. However, high concentrations of all such peptides were required to achieve effective inhibition of IgE binding. These high concentrations are predictive of excessively large doses for significant physiological effect, and are not therapeutically practical.
Anti-IgE antibodies have also been applied as a method for mapping sites on IgE that participate in binding to the Fcxcex5RI receptor. Studies with mouse monoclonal antibodies directed against various domains of IgE Fc revealed that anti-IgE monoclonal antibodies with specificities for the CH3 domain inhibit the binding of IgE to its high affinity receptor (Baniyash et al., Molec Immunol, 1988; 25: 705-711; and, Stadler et al., Immunol Cell Biol, 1996; 74: 195-200). These monoclonal antibody studies are in agreement with earlier studies that used polyclonal antipeptide antibodies to map sites that are apparently involved in receptor binding. For example, rabbit antibodies with specificities for IgE amino acid positions 401-415 (Burt et al., Molec Immunol, 1987; 24: 379-389), and 355-368 (Robertson and Liu, Molec Immunol, 1988; 25:103-113) showed specificity for unbound IgE but reacted poorly with receptor-bound IgE.
A canine IgE peptide fragment containing at least five continuous amino acids from dog IgE amino acids 356-479 is useful for the preparation of antibodies for diagnosis of allergy in dogs (JP 9179795, 1997). Those results are suggestive of surface-exposed effector sites in the CH3 domain of the dog xcex5 chain, but no such effector site is taught nor is a therapeutic application disclosed for the anti-IgE antibodies.
These epitope mapping studies demonstrate most consistently that the CH3 domain of the xcex5 heavy chain can be targeted for interventions aimed at inhibiting the binding of IgE to basophils and mast cells. However, the various studies are quite inconsistent on precise locations for sites on CH3 that are most useful. Also, results from cross-inhibition studies on IgE, with site-specific antibodies (e.g., Burt et al., 1987) have frequently been over-interpreted to signify that they have defined a precise location for the Fcxcex5R1 binding site on the xcex5 chain. Interpretation of such cross-inhibition studies is limited because it cannot be assumed that an antibody recognition site is equivalent to a ligand binding site. Antibodies may inhibit by directly binding to the desired target site, but they can also occupy non-continuous effector sites and inhibit ligand binding through steric hindrance or induction of conformational change.
Therefore, the epitope mapping studies have provided empirical observations but have not resolved the binding site for the high affinity receptor within the CH3 domain. In the absence of a defined binding site, no means is available for the reliable prediction of potentially therapeutic synthetic immunogens with immunologic crossreactivities for effector sites that participate directly or indirectly in binding to Fcxcex5R1.
Furthermore, in the absence of X-ray crystallography data for IgE, the available structural models for IgE are not sufficient for the reliable prediction of the sites on IgE that are suitable for anti-IgE interventions. Conflicting structures based on the divulged three-dimensional structure of IgG have been modeled for IgE and for the CH2/CH3 region of IgE that is associated with the interaction between IgE and its high affinity receptor. These models propose various conformationally dependent structures for the site, involving contact with linearly non-adjacent residues of the IgE molecule. Some models for the site suggest interactions between non-contiguous sites on the same xcex5 chain mediated by intramolecular disulfide bonded loops (Helm et al., Eur J Immunol, 1991; 21:1543-1548) or intramolecular loops maintained by electrostatic interactions (Presta et al., J Biol Chem, 1994; 269: 26368-26373). Other models propose intermolecular interactions between segments of the dimerized xcex5 chains of an IgE molecule (McDonnell et al., Biochem Soc Trans, 1997; 25: 387-392). In fact, experimental observations show that potential contact points comprise several scattered and discontinuous sites on the CH3 domain of the xcex5 chain and make it clear that the three-dimensional structure of the Fcxcex5R1 binding site cannot be readily resolved by modeling (Helm et al., 1988; Baniyash et al., 1988; and, Presta et al., 1994). Therefore, the identification of useful synthetic peptide antagonists and immunogens that mimic effector sites on IgE has not been disclosed by theoretical modeling. In the absence of a structure for IgE resolved by X-ray crystallography, such useful peptide sites can only be arrived at by empirical experimentation.
The concept of treating allergic diseases with anti-IgE antibodies, of specificities that inhibit the binding of IgE to the high affinity receptor on basophils and mast cells, also has been known (Stadler et al., 1996; Davis et al., 1993). Such anti-IgE antibodies are either anaphylactogenic (crosslinking) or non-anaphylactogenic (non-crosslinking). Most such anti-IgE antibodies are anaphylactogenic. They will bind and crosslink IgE on the surface of basophils and mast cells and trigger the release of the pharmacologic mediators of allergy. This crosslinking-could lead to anaphylaxis and death.
It is therefore crucial that anti-IgE antibodies for treatment be non-anaphylactogenic. Certain non-anaphylactogenic antibodies retain specificity for the CH3 domain of the c chain and do not crosslink cell-bound IgE, while displaying inhibitory activity for IgE-mediated histamine release (Davis et al., 1993; Stadler et al., 1996). Rup and Kahn (U.S. Pat. No. 4,940,782) report such a non-anaphylactogenic monoclonal antibody that reacts with free rat IgE and rat IgE bound to B cells, but not IgE bound to the rat mast cell Fcxcex5R1 receptor. Most significantly, it inhibits the sensitization of rat mast cells. The non-anaphylactogenic antibodies with homologous specificities for human IgE also inhibit sensitization by the same action mode. These anti-human IgE antibodies bind free serum IgE, bind to B cell-bound IgE, fail to bind to IgE attached to the basophil and mast cell high affinity receptor and prevent sensitization of human cells. These antibodies are presumed to act by specificity for the site on IgE that binds to the Fcxcex5R1 receptor (Rup and Kahn, U.S. Pat. No. 4,940,782; Davis et al., 1993; Chang, U.S. Pat. No. 5,420,251; Presta et al., J Immunol, 1993; 151: 2623-2632). In addition, a non-anaphylactogenic anti-human IgE monoclonal antibody with a different specificity has been found that also neutralizes free IgE (Rudolf et al., J Immunol, 1996; 157: 5646-5652). This anti-IgE does not directly bind with the receptor binding site because it also recognizes Fcxcex5R1-bound IgE. Apparently, it functions to reduce sensitization of basophils by altering the thermodynamic balance of receptor-bound versus free IgE.
Thus, anti-IgE antibodies that directly bind to the Fcxcex5R1 binding site and anti-IgE antibodies that interfere with Fcxcex5R1 binding at other effector sites, both serve to block the sensitization of mast cells and basophils by free IgE. These potentially immunotherapeutic antibodies identify CH3 as the domain of IgE that interacts with the high affinity IgE Fc receptor, in agreement with the previous mapping studies. However, a more precise identification of the binding site and alternative useful effector sites such as that described by Rudolf et al. remain elusive. Rudolf et al. have also used a phage display library to identify mimotope peptides which bind to their anti-IgE monoclonal antibody; however, the peptide mimotopes did not show homology to the primary amino acid sequence of human IgE (Rudolf et al., J. Immunol., 1998; 160: 3315-3321).
A humanized monoclonal anti-IgE antibody with apparent specificity for the Fcxcex5R1 receptor site is under clinical study in humans for the treatment of allergy by passive immunotherapy (MacGlashan et al., J Immunol, 1997; 158:1438-1445). It has been found that infusion with that antibody, rhuMAb-E25, reduces the serum concentration of IgE in patients, down-regulates the expression of IgE receptor on effector cells, reduces allergic sensitivities to challenge by allergen, and improves the symptoms of asthma and allergic rhinitis. The antibody displays a good safety profile. The clinical trial results establish the feasibility of an anti-IgE approach for the treatment of allergic diseases. But this treatment mode is problematical: Immunotherapy by the anti-IgE invention is accomplished by passive immunization, i.e., by infusion of the antibody. The antibody must be delivered in doses high enough and at frequencies often enough, via inconvenient intravenous or subcutaneous routes, to achieve a continuous pharmacologically effective concentration of antibody. The effective dose is determined by patient body weight, baseline level of free IgE in circulation, and by route of administration. In recent clinical trials, the steady-state concentration required for therapeutic efficacy was achieved by two weekly doses and maintained thereafter by biweekly doses. A full course of treatment for a typical allergy patient would expend a total of 2000-3000 mg of humanized antibody and requires seven to 10 inconvenient intravenous administrations (MacGlashan et al.,1997; Boulet et al., Am J Respir Crit Care Med, 1997; 155:1835-1840). The cost for this amount of antibody and the expense and inconvenience of multiple infusions in a hospital setting suggest that this treatment is too expensive for all but a small proportion of the patient population.
The clinical effectiveness of the monoclonal antibody rhuMAb-E25 establishes the feasibility of immunotherapy by passively administered anti-IgE. It also provides the rationale for an alternative anti-IgE approach by active immunization, if and when such immunogens can be designed.
An anti-IgE treatment affected by active immunization with an IgE immunogen, i.e., by xe2x80x9cvaccinationxe2x80x9d against endogenous IgE, would be preferable on the basis of cost and convenience. xe2x80x9cVaccinationxe2x80x9d against IgE offers advantages over passive immunization: small amounts of inexpensive immunogen, infrequent and conveniently administered intramuscular injections, and no need to customize murine antibodies for compatibility with the subject species, i.e., to xe2x80x9chumanizexe2x80x9d antibodies for use in humans, since the procedure uses the patient""s own immune system to produce antibodies. However, while the desensitizing monoclonal antibodies cited above may be suggestive of the desirability of IgE immunogens, they do not disclose the identity of safe and effective immunogens. Such immunogens must mimic relevant IgE effector sites with fidelity sufficient to evoke cross-inhibitory antibodies, while retaining site-specificity sufficient to avoid induction of anaphylactogenic antibodies. Moreover, effective IgE immunogens must be highly immunostimulatory. There remains a need for such immunogens, of relevant and safe site-specificity, and of sufficient immunopotency.
IgE immunogens for immunotherapy of allergy must be immunostimulatory so as to evoke levels of anti-IgE sufficient to reduce IgE-mediated sensitization. Such immunogens must be designed to overcome the strong tolerance exhibited towards self molecules. Haba and Nisonoff (Proc Natl Acad Sci USA, 1990; 87:3363-3367) induced an effective anti-IgE response in mice only by immunizations with IgE during a short neonatal window of development, from birth to day 10. Vaccinations initiated beyond this time failed to induce the desired autoimmune response unless the IgE used to immunize the mice had been covalently coupled to a foreign carrier protein, keyhole limpet hemocyanin (KLH). Similarly, a desensitizing anti-IgE response was evoked in rats by a recombinant protein comprising the CH2-CH3 xcex5 chain domains fused to the glutathione-S-transferase protein of Schistosoma japonicum (Hellman, Eur J Immunol, 1994; 24:415-420).
Other investigators have been concerned with minimizing the risk of evoking anaphylactogenic anti-IgE antibodies that crosslink IgE already bound to the surface of mast cells and basophils by seeking peptide IgE immunogens of finer site specificity. For example, a peptide corresponding to a site in the CH4 domain of IgE (residues 497-506 of SEQ ID NO:1) was coupled to KLH and used to induce polyclonal antibodies that were effective in suppressing IgE-mediated signal transduction in rat mast cells. However, the peptide-KLH conjugate displayed poor immunostimulatory capabilities which necessitated demonstration of efficacy by passive immunization of rats with peak immune rabbit antiserum (Stanworth et al., Lancet, 1990; 336:1279-1281). The CH4 immunogen of Stanworth et al. was later produced, by the work of the present inventor, as a series of wholly synthetic immunogens by synthesis that provided covalent linkage to promiscuous human T helper epitopes. Immunogenicity of these peptides was improved over that of the original KLH-peptide conjugate, but no evidence was provided for the efficacy of resultant anti-IgE CH4 antibodies (Wang, WO 95/26365). Furthermore, as shown herein in Example 1 (Table 2, entry 34), anti-peptide antibodies with specificity for the previously disclosed CH4 effector site (Stanworth et al., 1990) had no crossreactivity to human IgE. The earlier antipeptide studies of Burt and Stanworth (1987) targeted to the IgE-CH3 401-415 peptide also provided evidence of evoking desensitizing cross-reactivity, but this too required selected peak rabbit antiserum and use of an ill-defined peptide-carrier protein conjugate to observe effects by passive immunization in a rat model. No synthetic peptides have ever been demonstrated to be effective in eliciting the production in immunized hosts of polyclonal antisera capable of inhibition of histamine release.
The improvement of the prior art immunogens discussed above is necessary before a synthetic peptide immunogen of immunogenicity and specificity sufficient for efficacy and safety can be attained. The present invention accomplishes these improvements through incorporation of a collection of additional methods for the identification and design of synthetic peptide immunogens. These methods include: (1) an effective procedure for the identification of an effective target epitope; (2) the means to augment the immunogenicity of a B cell target epitope by combining it with a peptide comprising broadly reactive promiscuous T helper cell (Th) epitope; (3) the means of enlarging the repertoire of T cell epitopes by application of combinatorial peptide chemistry and thereby further accommodate the variable immune responsiveness of an outbred population; and (4) the stabilization of conformational features by the introduction of cyclic constraints, so as to maximize cross-reactivity to the native molecule.
Synthetic peptides have been used for xe2x80x9cepitope mappingxe2x80x9d to identify immunodominant determinants or epitopes on the surface of proteins, for the development of new vaccines and diagnostics. Epitope mapping employs a series of overlapping peptides corresponding to regions on the protein of interest to identify sites which participate in antibody-immunogen determinant interaction. Most commonly, epitope mapping employs peptides of relatively short length to precisely detect linear determinants. A fast method of epitope mapping known under the trademark xe2x80x9cPEPSCANxe2x80x9d is based on the simultaneous synthesis of hundreds of overlapping peptides, of lengths of 8 to 14 amino acids, coupled to solid supports. The coupled peptides are tested for their ability to bind antibodies. The PEPSCAN approach is effective in localizing linear determinants, but not for the identification of epitopes needed for mimicry of discontinuous effector sites such as the Fcxcex5R1 binding site (Meloen et al., Ann Biol Clin, 1991; 49:231-242). An alternative method relies on a set of nested and overlapping peptides of multiple lengths ranging from 15 to 60 residues. These longer peptides can be reliably synthesized by a laborious series of independent solid-phase peptide syntheses, rather than by the rapid and simultaneous PEPSCAN syntheses. The resulting set of long nested and overlapping peptides can then be used for analyses of antibody binding in systems such as experimental immunizations and natural infections, to identify long peptides which best present immunodominant determinants, including simple discontinuous epitopes. This method is exemplified by the studies of Wang for the mapping of immunodominant sites from HTLV I/II (U.S. Pat. No. 5,476,765) and HCV (U.S. Pat. No. 5,106,726); and it was used for the selection of a precise position on the gp120 sequence for optimum presentation of an HIV neutralizing epitope (Wang et al., Science, 1991; 254:285-288).
Peptide immunogens are generally more flexible than proteins and tend not to retain any preferred structure. Therefore it is useful to stabilize a peptide immunogen by the introduction of cyclic constraints. A correctly cyclized peptide immunogen can mimic and preserve the conformation of a targeted epitope and thereby evoke antibodies with cross-reactivities for that site on the authentic molecule (Moore, Chapter 2 in Synthetic Peptides: A User""s Guide, ed Grant, WH Freeman and Company: New York, 1992, pp 63-67).
Another important factor affecting the immunogenicity of an IgE-derived peptide for an allergy pharmaceutical is its presentation to the immune system by T helper cell epitopes that react with-a host""s T-helper cell receptors and Class II MHC molecules (Babbitt et al., Nature, 1985; 317: 359-361). These are often provided by carrier proteins with concomitant disadvantages due to the difficulties for the manufacture of well-defined peptide-carrier conjugates, misdirection of most antibody response to the carrier, and carrier-induced epitopic suppression (Cease, Intern Rev Immunol., 1990; 7: 85-107; Schutze et al., J Immunol., 1985; 135: 2319-2322). Alternatively, T-helper cell epitopes (Th) may also be supplied by synthetic peptides comprising Th sites. Thus, Th epitopes termed promiscuous Th evoke efficient T cell help and can be combined with synthetic B cell epitopes that by themselves are poorly immunogenic to generate potent peptide immunogens (U.S. Pat. No. 5,759,551). Well-designed promiscuous Th/B cell epitope chimeric peptides are capable of eliciting Th responses and resultant antibody responses in most members of a genetically diverse population expressing diverse MHC haplotypes. Promiscuous Th can be provided by specific sequences derived from potent foreign antigens, such as for example measles virus F protein, hepatitis B virus surface antigen, and Chlamydia trachomatis major outer membrane protein (MOMP). Many known promiscuous Th, taken from viral and bacterial pathogens, have been shown to be effective in potentiating a poorly immunogenic peptide corresponding to the decapeptide hormone LHRH (U.S. Pat. No. 5,759,551)
Promiscuous Th epitopes derived from foreign pathogens may include, but are not limited to, hepatitis B surface and core antigen helper T cell epitopes (HBs Th and HBc Th), pertussis toxin helper T cell epitopes (PT Th), tetanus toxin helper T cell epitopes (TT Th), measles virus F protein helper T cell epitopes (MVF Th), Chlamydia trachomatis major outer membrane protein helper T cell epitopes (CT Th), diphtheria toxin helper T cell epitopes (DT Th), Plasmodium falciparum circumsporozoite helper T cell epitopes (PF Th), Schistosoma mansoni triose phosphate isomerase helper T cell epitopes (SM Th), and Escherichia coli TraT helper T cell epitopes (TraT Th). The pathogen-derived Th were listed as SEQ ID NOS:2-9 and 42-52 in U.S. Pat. No. 5,759,551; as Chlamydia helper site P11 in Stagg et al., Immunology, 1993; 79;1-9; and as HBc peptide 50-69 in Ferrari et al., J Clin Invest, 1991; 88: 214-222.
Promiscuous Th epitopes range in size from about 15 to about 50 amino acid residues in length (U.S. Pat. No. 5,759,551) and often share common structural features and may contain specific landmark sequences. For example, a common feature is amphipathic helices, which are alpha-helical structures with hydrophobic amino acid residues dominating one face of the helix and with charged and polar resides dominating the surrounding faces (Cease et al., Proc Natl Acad Sci USA, 1987; 84:4249-4253). Th epitopes frequently contain additional primary amino acid patterns such as a Gly or charged residue followed by two to three hydrophobic residues, followed in turn by a charged or polar residue. This pattern defines what are called Rothbard sequences. Also, Th epitopes often obey the 1, 4, 5, 8 rule, where a positively charged residue is followed by hydrophobic residues at the fourth, fifth and eighth positions after the charged residue, consistent with an amphipathic helix having positions 1, 4, 5 and 8 located on the same face. Since all of these structures are composed of common hydrophobic, charged and polar amino acids, each structure can exist simultaneously within a single Th epitope (Partidos et al., J Gen Virol, 1991; 72:1293-1299). Most, if not all, of the promiscuous T cell epitopes fit at least one of the periodicities described above. These features may be incorporated into the designs of xe2x80x9cidealized artificial Th sitesxe2x80x9d.
Useful Th sites may also include combinatorial Th that incorporate selected degenerate sites into the design of the idealized Th sites. In Wang et al. (WO 95/11998), a particular class of a combinatorial epitope was designated as a xe2x80x9cStructured Synthetic Antigen Libraryxe2x80x9d or SSAL. A Th constructed as an. SSAL epitope is composed of positional substitutions organized around a structural framework of invariant residues. The sequence of the SSAL is determined by aligning the primary amino acid sequence of a promiscuous Th, retaining relatively invariant residues at positions responsible for the unique structure of the Th peptide and providing degeneracy at the positions associated with recognition of the diverse MHC restriction elements. Lists of variable and preferred amino acids are available for MHC-binding motifs (Meister et al., Vaccine, 1995; 13: 581-591: Alexander et al., Immunity, 1994, 1:751-761).
All members of the SSAL are produced simultaneously in a single solid-phase peptide synthesis in tandem with the targeted B cell epitope and other sequences. The Th library sequence maintains the binding motifs of a promiscuous Th and accommodates reactivity to a wider range of haplotypes. For example, the degenerate Th epitope described in WO 95/11998 as xe2x80x9cSSAL1TH1xe2x80x9d was modeled after a promiscuous epitope taken from the F protein of measles virus (Partidos et al., 1991). SSAL1TH1 was designed to be used in tandem with an LHRH target peptide. Like the measles epitope, SSAL1TH1 follows the Rothbard sequence and the 1, 4, 5, 8 rule:
Charged residues Glu or Asp are added at position 1 to increase the charge surrounding the hydrophobic face of the Th. The hydrophobic face of the amphipathic helix is then maintained by hydrophobic residues at 2, 5, 8, 9, 10, 13 and 16, with variability at 2, 5, 8, 9, 10, 13, and 16 to provide a facade with the capability of binding to a wide range of MHC restriction elements. The net effect of the SSAL feature is to enlarge the range of immune responsiveness to an artificial Th (WO 95/11998).
Peptide immunogens that have been designed with the peptide technologies and peptide design elements discussed above, i.e., precise epitope mapping, cyclic constraint, and the incorporation of promiscuous Th epitopes or idealized promiscuous Th, and idealized SSAL Th epitopes, are the basis for the effective synthetic peptide IgE immunogens of the present invention. Such peptides are preferred for appropriate targeting and safety due to effective presentation of the IgE effector site by optimized positioning and cyclization, and for immunopotency due to broadly reactive Th responsiveness.
The present invention provides new synthetic peptide conjugate compositions for the treatment of IgE-mediated allergic diseases by active immunization. The immunization induces the production of high titer non-anaphylactogenic polyclonal antibodies specific to an effector site of IgE in an immunized host. This in turn prevents the triggering and activation of mast cells/basophils and down-regulates IgE synthesis.
Treatment is effected by immunization of the host with the peptide composition, with each peptide contained therein comprising a target antigenic peptide sequence (referred to herein as an xe2x80x9cIgE-CH3 domain antigenxe2x80x9d or xe2x80x9cIgE-CH3 domain antigen-peptidexe2x80x9d) modified from a segment of the CH3 domain of the epsilon (xcex5) heavy chain of human IgE (e.g., amino acids 413-435 of SEQ ID No:1 or SEQ ID NO:5) or the homologous sequence from other species (e.g. SEQ ID NOS:6-8 and 84).
In general, the IgE-CH3 domain antigen is a peptide sequence between about 25 and about 29 amino acids in length, is substantially homologous to the above segment of the CH3 domain of the epsilon heavy chain of a mammalian IgE antibody, and contains two cysteine residues separated by about 23 amino acid residues. In the present context, substantially homologous means that in addition to the two cysteine residues, which may be introduced by insertion or substitution, up to about four other amino acid substitutions (preferably conservative substitutions) may also be made.
Preferably, the target site is modified from that of the naturally occurring IgE sequences as follows:
(1) by the insertion of a cysteine residue to the N-terminus side of position 413 or homologous position, unless cysteine is already present at positions 413 or 414 in the natural sequence;
(2) by the conservative substitution (preferably of serine) for any native cysteines from positions 415 to 434 of the natural target sequence;
(3) by the insertion of cysteine at the C-terminus side of position 435 or homologous position unless cysteine is already present at positions 435 or 436 in the natural sequence; and
(4) by the formation of a disulfide bond between the retained cysteines so as to produce a cyclic structure. The structures may also comprise 1 to 5 additional amino acids taken from either terminus of the 413-435 segment of IgE, provided that the single disulfide looped structure is preserved.
An optimized IgE-CH3 domain antigen peptide for human IgE, having the sequence Cys-Gly-Glu-Thr-Tyr-Gln-Ser-Arg-Val-Thr-His-Pro-His-Leu-Pro-Arg-Ala-Leu-Met-Arg-Ser-Thr-Thr-Lys-Cys (SEQ ID NO:5) is provided by the present invention. The human IgE target site is cyclized through the unnatural terminal cysteines and a serine residue substitutes for the cysteine residue of the natural sequence. Antibody that is evoked by peptide immunogens comprising this IgE-CH3 domain antigen is crossreactive with human IgE and inhibits the sensitization of human basophils by human IgE.
Likewise, corresponding target sites for IgE of other species can be derived from the homologous xcex5 chain segment of the relevant species. For example, such target sequences can be taken from the dog, rat and mouse xcex5 sequences shown in Table 1 (SEQ ID NOS: 2, 3 and 4), or the horse IgE-CH3 sequence provided by Navarro et al., Molec. Immunol., 1995, 32:1-8. Additional IgE-CH3 domain antigen peptides (SEQ ID NOS: 6, 7, B, and 84), may be derived from these sequences.
Preferably, the IgE-CH3 domain antigens of the invention are rendered more immunogenic via covalent linkage to a carrier protein through chemical coupling, or more preferably via covalent linkage to synthetic immunostimulatory elements, such as promiscuous Th epitopes, through direct synthesis. Specific examples of carrier protein and immunostimulatory elements are provided, e.g., Keyhole Limpet Hemocyanin (KLH) carrier, an artificial Th (SEQ ID NO:9), artificial SSAL Th (SEQ ID NOS:10 and 11), a pathogen-derived Th (SEQ ID NO:12), and an immunostimulatory invasin peptide (Inv) taken from Yersinia (SEQ ID NO:13).
Completely synthetic peptide conjugates of the invention may be represented by the formulas:
(A)n-(IgE-CH3 domain antigen)-(B)o-(Th)m-X.
or
(A)n-(Th)m-(B)o-(IgE-CH3 domain antigen)-X
or
(A)n-(B)o-(Th)m-(B)o-(IgE-CH3 domain antigen)-X
or
(IgE-CH3 domain antigen)-(B)o-(Th)m-(A)n-X
xe2x80x83or
(Th)m-(B)o-(IgE-CH3 domain antigen)-(A)n-X
wherein
each A is independently an amino acid or a general immunostimulatory sequence;
each B is chosen from the group consisting of amino acids,
xe2x80x94NHCH(X)CH2SCH2COxe2x80x94, xe2x80x94NHCH(X)CH2SCH2CO(xcex5-N)Lys-, xe2x80x94NHCH(X)CH2S-succinimidyl(xcex5-N)Lys-, and xe2x80x94NHCH(X)CH2S-(succinimidyl)-;
each Th is independently a sequence of amino acids that constitutes a helper T cell epitope, or an immune enhancing analog or segment thereof;
IgE-CH3 domain antigen is a peptide between about 25 and about 29 amino acids in length, is substantially homologous to one of the segments represented by SEQ ID NOS:5-8 and 84 of the CH3 domain of the epsilon heavy chain of a mammalian IgE antibody, and contains two cysteine residues separated by about 23 amino acid residues;
X is an amino acid xcex1-COOH or xcex1-CONH2;
n is from 0 to about 10;
m is from 1 to about 4; and
o is from 0 to about 10.
More specifically, IgE-CH3 domain antigen is selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, homologous sequences from the epsilon heavy chain of mammalian IgE-CH3 antibodies, and crossreactive and immunologically functional analogs thereof.
The peptide compositions of the present invention comprises peptide immunogens from about 25 to about 100 amino acid residues, preferably from about 25 to about 80 amino acid residues and more preferably from about 45 to about 65 amino acid residues.
Also provided are adjuvants and/or delivery vehicles and other ingredients routinely incorporated with vaccine formulations, and instructions for dosage such that immunotherapeutic antibodies directed against the targeted IgE effector site are generated. This in turn inhibits the sensitization by circulatory IgE of basophils and mast cells, and thereby prevents the triggering and activation of mast cells/basophils by IgE-allergen complexes. The inhibitory mechanism, mediated by the antibodies and induced by the peptide composition of the present invention, will specifically reduce or eliminate the IgE-mediated pathology while leaving the defensive components of the immune system, e.g. IgG, unaffected.